U.S. patent application number 12/498102 was filed with the patent office on 2010-01-14 for induction of gene expression using a high concentration sugar mixture.
Invention is credited to George R. England, Aaron Kelley, Colin Mitchinson.
Application Number | 20100009408 12/498102 |
Document ID | / |
Family ID | 32107864 |
Filed Date | 2010-01-14 |
United States Patent
Application |
20100009408 |
Kind Code |
A1 |
England; George R. ; et
al. |
January 14, 2010 |
Induction of Gene Expression Using a High Concentration Sugar
Mixture
Abstract
Described herein is a composition useful for inducing expression
of genes whose expression is under control of an inducible promoter
sequence and methods for the compositions preparation and use.
Inventors: |
England; George R.; (Redwood
City, CA) ; Kelley; Aaron; (Mountain View, CA)
; Mitchinson; Colin; (Half Moon Bay, CA) |
Correspondence
Address: |
Victoria L. Boyd;Genencor International, Inc.
925 Page Mill Road
Palo Alto
CA
94304-1013
US
|
Family ID: |
32107864 |
Appl. No.: |
12/498102 |
Filed: |
July 6, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10660123 |
Sep 10, 2003 |
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12498102 |
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60409466 |
Sep 10, 2002 |
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Current U.S.
Class: |
435/69.4 ;
435/209; 435/69.1; 435/69.6; 435/71.1; 435/71.2 |
Current CPC
Class: |
C12P 19/14 20130101;
C12N 9/2445 20130101; C12N 9/2437 20130101; C12N 1/38 20130101;
C12P 19/12 20130101; A61P 21/02 20180101; C12N 1/14 20130101; C12P
21/02 20130101; C12Y 302/01021 20130101 |
Class at
Publication: |
435/69.4 ;
435/209; 435/71.1; 435/69.1; 435/69.6; 435/71.2 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C12N 9/42 20060101 C12N009/42 |
Goverment Interests
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Portions of this work were funded by Subcontract No.
ZCO-0-30017-01 with the National Renewable Energy Laboratory under
Prime Contract No. DE-AC36-99GO10337 with the U.S. Department of
Energy. Accordingly, the United States Government may have certain
rights in this invention.
Claims
1. A method of producing an inducing feed composition, said method
comprising the steps of: a. Mixing a first solution with a whole
cellulase preparation to give a first mixture; and b. Incubating
the first mixture at a temperature and for a sufficient time to
produce the inducing feed composition.
2. The method of claim 1 wherein the first solution is a
concentrated glucose solution comprising from about 5% to about 75%
(wt/wt) glucose.
3. The method of claim 1 wherein the first solution is a
concentrated glucose solution comprising from about 50% to about
75% (wt/wt) glucose.
4. The method of claim 1 wherein the first solution is a cellobiose
solution comprising from about 5% to about 40% (wt/wt)
cellobiose.
5. The method of claim 1 wherein the first solution is a cellobiose
solution comprising from about 20% to about 40% (wt/wt)
cellobiose.
6. The method of claim 1 wherein the whole cellulase preparation is
from about 2 g/L to about 10 g/L protein.
7. The method of claim 1 wherein the whole cellulase preparation is
about 5 g/L protein.
8. The method of claim 1 wherein the temperature is from about
50.degree. C. to about 75.degree. C.
9. The method of claim 1 wherein the solution is incubated for
between 8 hours and 500 hours.
10. The method of claim 1 wherein the solution is incubated for
between 48 hours and 72 hours.
11. An inducing feed composition produced by the method of claim
1.
12. The inducing feed composition of claim 11 comprising a mixture
of sugars.
13. The inducing feed composition of claim 11 comprising
sophorose.
14. The inducing feed composition of claim 11 comprising
gentiobiose.
15. A method for producing proteins comprising providing a host
cell with the inducing feed composition of claim 11.
16. The method of claim 15 wherein the protein produced is an
endogenous cellulase.
17. The method of claim 15 wherein the host cell has been
transformed with an expression construct comprising a promoter
operably linked to a gene encoding a protein of interest.
18. The method of claim 17 wherein the promoter is an inducible
promoter.
19. The method of claim 17 wherein the promoter is a cellulase gene
promoter.
20. The method of claim 19 wherein the promoter is the cbh 1
promoter from Trichoderma reesei.
21. The method of claim 18 wherein the inducible promoter is a
sophorose-inducible promoter.
22. The method of claim 18 wherein the inducible promoter is a
gentiobiose-inducible promoter.
23. The method of claim 17 wherein the protein of interest is a
heterologous protein.
24. The method of claim 23 wherein the heterologous protein is
selected from the group consisting of hormones, enzymes, growth
factors, cytokines, and antibodies.
25. The method of claim 15 wherein the host cell is a filamentous
fungus.
26. The method of claim 25 wherein the fungus is selected from the
group consisting of Trichoderma, Humicola, Fusarium, Aspergillus,
Neurospora, Penicillium, Cephalosporium, Achlya, Podospora,
Endothia, Mucor, Cochliobolus and Pyricularia.
27. The method of claim 26 wherein the fungus is Trichoderma
spp.
28. The method of claim 27 wherein the fungus is Trichoderma
reesei.
29. The method of claim 26 wherein the fungus is Penicillium
spp.
30. The method of claim 29 wherein the fungus is Penicillium
funiculosum.
31. The method of claim 15 wherein the host cell is a bacteria.
32. The method of claim 31 wherein the bacteria is selected from
the group consisting of Streptomyces, Thermomonospora, Bacillus,
and Cellulomonas.
33. The method of claim 1 wherein the whole cellulase preparation
is immobilized
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 60/409,466, filed Sep. 10, 2002 (Attorney Docket
No. GC774P), which is herein incorporated in its entirety by
reference.
FIELD OF THE INVENTION
[0003] This invention relates to methods for improved production of
proteins from a cell culture. The inventors have discovered culture
components and conditions that dramatically increase the amount of
protein produced from genes under the control of cellulase gene
promoter sequences. The improved methods can be used for the
production of proteins encoded by naturally occurring cellulase
genes as well as from various heterologous constructs.
BACKGROUND OF THE INVENTION
[0004] Filamentous fungi and cellulolytic bacteria produce
extracellular cellulase enzymes that confer on the organisms the
ability to hydrolyze the .beta.-(1,4)-linked glycosidic bonds of
cellulose to produce glucose. These enzymes provide the organisms
with the ability to use cellulose, the most abundant plant
polysaccharide, for growth.
[0005] The filamentous fungus, Trichoderma reesei, is an efficient
producer of cellulase enzymes. As such Trichoderma reesei has been
exploited for its ability to produce these enzymes, which are
valuable in the production of such commodities as fuel ethanol,
clothing, detergents, fibers and other products.
[0006] The cellulolytic mix of Trichoderma reesei proteins is among
the best characterized cellulolytic pathways of microorganisms. The
cellulases that comprise these mixes are classified into two broad
categories: the endoglucanases (EG) and the cellobiohydrolases
(CBH). .beta.-glucosidase is also part of the cellulase mix of
Trichoderma reesei.
[0007] Trichoderma reesei has also been exploited for its ability
to produce heterologous proteins. Genes encoding a desired protein
can be regulated when they are operably linked to the inducible
cbh1 promoter of T. reesei. Foreign polypeptides have been secreted
in Trichoderma reesei as fusions with the catalytic domain plus
linker region of cbh1 (Nyyssonen et al., Bio/technology 11:591-595,
1993).
[0008] Expression of the genes comprising the cellulase system is
coordinate and regulated at the transcriptional level. The members
of this system act synergistically, and as noted above, are
necessary for the efficient hydrolysis of cellulose to soluble
oligosaccharides.
[0009] Expression and production of the main cellulase genes in
Trichoderma, cbh1, cbh2, egl1, and egl2, is dependent on the carbon
source available for growth. The cellulase genes are tightly
repressed by glucose and are induced several thousand fold by
cellulose or the disaccharide sophorose. Indeed, the expression
level of the major cellobiohydrolase 1 (cbh1) is up-regulated
several thousand fold on media containing inducing carbon sources
such as cellulose or sophorose compared with glucose containing
media (Ilmen et al., App. Environ. Microbio., 1298-1306,1997).
[0010] Commercial scale production of cellulase enzymes is by
either solid or submerged culture including batch, fed batch, and
continuous flow processes. The most problematic and expensive
aspect of industrial cellulase production is providing the
appropriate inducer to Trichoderma. As is the case for laboratory
scale experiments, cellulase production on a commercial scale is
induced by growing the fungus on solid cellulose or by culturing
the organism in the presence of a disaccharide inducer such as
lactose. Unfortunately on an industrial scale, both methods of
induction have drawbacks which result in high costs being
associated with cellulase production.
[0011] Cellulase synthesis is subject to both cellulose induction
and glucose repression. Thus, a critical factor influencing the
yield of cellulase enzymes or heterologous proteins under the
control of an inducible promoter is the maintenance of a proper
balance between cellulose substrate and glucose concentration; it
is critical for obtaining reasonable commercial yields of the
regulated gene product. Although cellulose is an effective and
inexpensive inducer, controlling the glucose concentration when
Trichoderma is grown on solid cellulose can be problematic. At low
concentrations of cellulose, glucose production may be too slow to
meet the metabolic needs of active cell growth and function. On the
other hand, cellulase synthesis can be halted by glucose repression
when glucose generation is faster than consumption. Thus, expensive
process control schemes are required to provide slow substrate
addition and monitoring of glucose concentration (Ju and Afolabi,
Biotechnol. Prog., 91-97, 1999). Moreover, the slow continuous
delivery of substrate can be difficult to achieve as a result of
the solid nature of the cellulosic materials.
[0012] Allen and Mortensen (Biotechnol. Bioeng., 2641-45,1981) have
shown that 200 IU/ml of purified .beta.-glucosidase from
Aspergillus phoenicis when incubated with a 50% glucose syrup
produces a solution with the ability to induce cellulase production
when used as a carbon source. Purification of the
.beta.-glucosidase is both time-consuming and expensive. In
addition, these authors used more than 20.times. the
.beta.-glucosidase loading compared to that used in this current
work.
[0013] Some of the problems associated with the use of cellulose as
an inducing substrate can be overcome through the use of soluble
substrates and inducers such as lactose or sophorose. Lactose has
to be provided at high concentrations so as to function as an
inducer and a carbon source. (See Seiboth, et. al., Mol. Genet.
Genomics, 124-32, 2002.) Gentiobiose may also serve as an inducer.
Sophorose is a more potent inducer than cellulose, but sophorose is
expensive and difficult to manufacture. Thus, while it is easier to
handle and control than solid cellulose, sophorose can nonetheless
make the cost of producing cellulases prohibitively expensive and,
thus, is impractical for commercial cellulase production. Clearly,
a need exists for a convenient, soluble substrate composition that
also provides an inexpensive method of cellulase induction in
filamentous fungi, e.g., Trichoderma reesei.
[0014] In addition, the ability to regulate inducible promoters to
express either endogenous or heterologous genes with an inexpensive
inducing agent would be of great commercial benefit.
BRIEF SUMMARY OF THE INVENTION
[0015] It has now been discovered that when a whole cellulase
preparation is added to a concentrated glucose solution, and the
composition is incubated for at least two days at 50.degree. C. to
about 65.degree. C., a sugar mixture containing appreciable
quantities of an inducer of cellulase gene expression is produced.
Surprisingly, the resulting complex mixture is sufficient to induce
cellulase production as is without further purification. This
discovery is surprising since glucose acts as a repressor of
cellulase genes in Trichoderma reesei. This discovery provides an
inducer of cellulase gene expression that is an inexpensive
alternative to lactose or purified sophorose and a less cumbersome
alternative to solid cellulose for the production of cellulases in
Trichoderma reesei.
[0016] In one embodiment the invention provides a composition for
inducing expression of genes whose expression is under control of
cellulase gene promoter sequences, comprising: (i) from about 5% to
about 75% (wt/wt) glucose, preferably 50%-70% glucose and (ii) from
about 2 g/L to about 10 g/L total protein, preferably 5 g/L of
whole cellulase preparation wherein the composition is incubated at
about 50.degree. C. to about 70.degree. C. for several days prior
to use to promote formation within the composition of an inducer of
gene expression.
[0017] In another embodiment the inducing feed composition is
incubated at about 50.degree. C. to about 65.degree. C., preferably
at about 55.degree. C. for 48 hours before use.
[0018] In another embodiment the inducing feed composition is
incubated at about 50.degree. C. to about 65.degree. C., preferably
at about 65.degree. C. for 72 hours before use.
[0019] In a preferred embodiment the incubation product that
results from incubating a concentrated glucose solution with whole
cellulase preparation, is a mixture of sugars containing sophorose.
In another preferred embodiment the incubation product is a mixture
of sugars containing gentiobiose.
[0020] In one embodiment the invention provides a method for
producing proteins whose gene expression is under control of an
inducible promoter sequence, wherein a cell culture is provided,
and an inducing feed composition resulting from incubation of a
whole cellulase preparation in a concentrated glucose solution is
added to the culture in an amount effective for inducing the
expression of genes under control of the inducible promoter
sequence.
[0021] The improved methods can be used for the production of
proteins encoded by naturally occurring cellulase genes as well as
from various heterologous constructs. Such constructs include
expression vectors wherein the gene encoding the protein of
interest is operably linked to an inducible promoter. In one
embodiment, the inducible promoter is a cellulase gene promoter. In
a second embodiment, the inducible promoter is a
sophorose-inducible promoter. In a third embodiment, the inducible
promoter is a gentiobiose-inducible promoter. In one aspect, the
inducible promoter is a cbh 1 promoter.
[0022] In an embodiment the method for producing a protein of
interest produces a protein selected from the group consisting of
hormones, enzymes, growth factors, cytokines, and antibodies. In
one aspect, the method is used to produce proteins that are
naturally occurring cellulase enzymes. In another aspect, the
method is used to produce proteins whose expression is not
naturally under control of cellulase gene promoter sequences.
[0023] In another embodiment, the method for producing proteins
employs a filamentous fungus. In one aspect the fungus is
Trichoderma. In another aspect, the fungus is Trichoderma
reesei.
[0024] In a further embodiment the method for producing a protein
of interest utilizes an inducing composition produced by adding a
whole cellulase preparation to a cellobiose solution, and the
cellulase-cellobiose solution is incubated for at least two days at
50.degree. C. to about 70.degree. C. to form an inducing feed
composition. In one aspect the solution is incubated for at least
two days at 50.degree. C. to about 65.degree. C. to form an
inducing feed composition. This composition is a sugar mixture
containing appreciable quantities of an inducer of cellulase gene
expression.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 illustrates the effects on wild-type T. reesei
(RLP-37) (see Sheir-Neiss and Montenecourt, Appl. Microbio.
Biotechnol., 46-53, 1984) cellulase production of feeding the
inventive inducing composition (.box-solid.; squares) compared with
a glucose composition (.diamond-solid.; diamonds).
[0026] FIG. 2 is a graph that illustrates the differences between
the production of sophorose by immobilized enzyme (.box-solid.;
squares) compared with an enzyme solution (.diamond-solid.;
diamonds). The final glucose concentration is approximately 40%.
The protein loading was 10 g/L. See example 4 for details.
[0027] FIG. 3 is a graph that illustrates the differences between
the production of sophorose by immobilized enzyme (.box-solid.;
squares) compared with an enzyme solution (.tangle-solidup.;
triangles). The final glucose concentration is approximately 60%.
The protein loading was 3.2 g/L. See example 4 for details.
[0028] FIG. 4 is a graph comparing the results from a second run of
the experiments with the first run of experiments described for
FIGS. 2 and 3 that used the same immobilized enzyme. The
immobilized enzyme recovered after the first run retained activity
in the second run. Symbols are: .box-solid. (squares), first run of
10 g/L experiment; .diamond-solid. (diamonds), second run of 10 g/L
experiment; .tangle-solidup. (triangles), first run of 3.2 g/L
experiment; X, second run of 3.2 g/L experiment.
[0029] FIG. 5 is a graph showing sophorose production in 25%
Cellobiose compared to 25% glucose. Sophorose production in 25%
cellobiose (.box-solid.; squares) or glucose solution (w/w) ( ;
circles).
[0030] FIG. 6 is a graph showing sophorose production in 60%
glucose solution (w/w) at different loadings of whole cellulase.
.tangle-solidup. (triangles), 2.5 g/L, .box-solid. (squares), 5.0
g/L, .diamond-solid. (diamonds), 7.5 g/L, X, 10 g/L whole
cellulase.
DETAILED DESCRIPTION
[0031] The filamentous fungus Trichoderma reesei is one of the most
extensively studied cellulolytic organisms (reviewed e.g. by
Nevalainen and Penttila, Mycota, 303-319, 1995). In industry, the
cellulolytic enzymes of Trichoderma are used for many purposes
including; production of fuel ethanol, paper, rayon, cellophane,
detergents and fibers. Cellulase enzymes are also used to improve
the nutritional value of animal feeds, and to facilitate the
extraction of valuable components from plant cells (Mandels,
Biochem. Soc. Trans., 414-16. 1985). Thus, these enzymes are of
primary importance in the production of many useful products.
[0032] The production of cellulases in Trichoderma is dependent on
the carbon source available. Cellulose, lactose and the
disaccharide sophorose, induce cellulase synthesis by Trichoderma
reesei. Conversely, the presence of glucose results in tight
repression of cellulase gene expression. Providing the appropriate
inducer for industrial scale production is a major problematic
factor contributing to high production costs of cellulase
enzymes.
[0033] It has now been discovered that when a whole cellulase
preparation is added to a concentrated glucose solution, and the
composition is incubated for at least two days at about 50.degree.
C. to about 75.degree. C., preferably about 50.degree. C. to
65.degree. C., a sugar mixture containing appreciable quantities of
an inducer of cellulase gene expression is made, i.e, the inducing
feed composition. The inducing feed composition has between about 2
and 25 g/L sophorose. In addition, the inducing feed composition
has between about 35 and 60 g/L gentiobiose. Surprisingly, the
resulting mixture does not need any further purification. It is
competent to induce cellulase production as is. This discovery
provides the inexpensive alternative to lactose or purified
sophorose that is needed by industry, as well as a less cumbersome
alternative to solid cellulose for the production of proteins
regulated by inducible promoters in a filamentous fungus. It is
specifically contemplated that the inventive composition is useful
for cellulase production in Trichoderma.
[0034] In an alternative method of producing the inducing feed
composition, the end fermentation broth (whole cellulase plus
cells) may be added to a glucose solution (e.g., 20%). The presence
of the cells does not affect sophorose formation. Thus, there is no
need to use a recovered cellulase (i.e., a cellulase preparation
isolated from the cells). The enzyme mixture present at the end of
a fermentation may be used although the cells are still
present.
[0035] In one embodiment, the invention provides a composition
comprising a concentrated glucose solution and whole cellulase
preparation that can be used as an inducing feed for the production
of a protein of interest by a filamentous fungus. In one aspect,
the protein of interest is a cellulolytic enzyme. In another
aspect, the protein of interest is a heterologous protein. In an
embodiment the inducing feed induces cellulase enzyme production by
Trichoderma reesei. It is surprising that the solution is effective
at inducing cellulase gene expression, since cellulase genes are
known to be repressed by the presence of glucose.
[0036] In one embodiment an inducing feed is made by preparing a
sterile solution of 5%-75% (wt/wt) glucose. A whole cellulase
preparation from Trichoderma reesei is added to a sterile glucose
solution to a final concentration of between 2 g and 20 g total
protein/L. The final protein range may be as low as 0.5 g/L and as
high as 50 g/L. In one aspect the .beta.-glucosidase activity in
the glucose solution is greater than 1.5 IU/ml. In one aspect the
.beta.-glucosidase activity in the glucose solution is less than
200 IU/ml. In another aspect .beta.-glucosidase activity of the
glucose solution is between 1.5 IU/ml and 200 IU/ml. In another
aspect .beta.-glucosidase activity of the glucose solution is
between 1.9 IU/ml and 200 IU/ml. In another aspect
.beta.-glucosidase activity of the glucose solution is between 9.3
IU/ml and 200 IU/ml. In another aspect .beta.-glucosidase activity
of the glucose solution is between 1.5 IU/ml and 180 IU/ml. In
another aspect .beta.-glucosidase activity of the glucose solution
is between 9.3 IU/ml and 180 IU/ml. The solution is incubated at
50.degree. C.-75.degree. C., preferably between 50.degree. C. and
65.degree. C. The solution is incubated for between 8 hours and 7
days with mixing. In one embodiment the incubation period is
greater than two days. In second embodiment the incubation period
is two days. In third embodiment the incubation period is three
days. The final sterile solution is harvested and used for
fermentation feeding. In one embodiment the inducing feed is
prepared with a 60% (wt/wt) glucose solution. In another embodiment
the inducing feed is prepared by adding whole cellulase preparation
to the glucose solution to a final concentration of 2 g total
protein /L.
[0037] Another object of the invention herein is to provide for the
expression and secretion by the host filamentous fungus of desired
proteins heterologous to said host filamentous fungus. The proteins
produced by the induction of genes whose expression is controlled
by an inducible promoter sequence include naturally occurring
cellulase proteins, as well as various heterologous proteins. In a
preferred embodiment, the protein expressed under control of
inducible promoter sequences is a hormone, enzyme, growth factor,
cytokine, or antibody.
[0038] Various species of filamentous fungi may be used as
expression hosts including the following genera: Aspergillus,
Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya,
Podospora, Endothia, Mucor, Cochliobolus and Pyricularia. Specific
expression hosts include Trichoderma reesei, e.g. NRRL 15709, ATCC
13631, 56764, 56765, 56466, 56767, Trichoderma viride, e.g., ATCC
32098 and 32086 Aspergillus nidulans, (Yelton, M., et al. (1984)
Proc. Natl. Acad. Sci. USA, 81,1470-1474; Mullaney, E. J. et al.
(1985) Mol. Gen. Genet. 199, 37-45; John, M. A. and J. F. Peberdy
(1984) Enzyme Microb. Technol. 6, 386-389; Tilburn, et al. (1982)
Gene 26,205-221; Ballance, D. J. et al., (1983) Biochem. Biophys.
Res. Comm. 112,284-289; Johnston, I. L. et al. (1985) EMBO J.4,
1307-1311) A. niger, (Kelly, J. M. and M. Hynes (1985) EMBO 4,
475-479) Aspergillus awamori, e.g., NRRL 3112, ATCC 22342, ATCC
44733, ATCC 14331 and strain UVK 143f, Aspergillus oryzae, e.g.,
ATCC 11490, and Neurospora crassa (Case, M. E. et al. (1979) Proc.
Natl. Acad. Scie. USA, 76, 5259-5263; Lambowitz U.S. Pat.
No.4,486,553; Kinsey, J. A. and J. A. Rambosek (1984) Molecular and
Cellular Biology 4,117-122; Bull, J. H. and J. C. Wooton (1984)
Nature 310, 701-704).
[0039] In a preferred embodiment, the microbial host is a member of
the species of Trichoderma, Humicola, Fusarium, Aspergillus,
Streptomyces, Thermomonospora, Bacillus, or Cellulomonas.
I. Definitions
[0040] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Typically, the antigen-binding region of an antibody or its
functional equivalent will be most critical in specificity and
affinity of binding. See Paul, Fundamental Immunology.
[0041] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (VL) and variable heavy chain (VH) refer to
these light and heavy chains respectively.
[0042] "Cellulase," "cellulolytic enzymes" or "cellulase enzymes"
means bacterial, or fungal exoglucanases or exocellobiohydrolases,
and/or endoglucanases, and/or .beta.-glucosidases. These three
different types of cellulase enzymes act synergistically to convert
cellulose and its derivatives to glucose.
[0043] Many microbes make enzymes that hydrolyze cellulose,
including the wood rotting fungus Trichoderma, the compost bacteria
Thermomonospora (now Thermobifida), Bacillus, and Cellulomonas;
Streptomyces; and the fungi Humicola, Aspergillus and Fusarium. The
enzymes made by these microbes are mixtures of proteins with three
types of actions useful in the conversion of cellulose to glucose:
endoglucanases (EG), cellobiohydrolases (CBH), and beta-glucosidase
(BG).
[0044] As used herein, the phrases "whole cellulase preparation"
and "whole cellulase composition" are used interchangeably and
refer to both naturally occurring and non-naturally occurring
compositions. A "naturally occurring" composition is one produced
by a naturally occurring source and which comprises one or more
cellobiohydrolase-type, one or more endoglucanase-type, and one or
more .beta.-glucosidase components wherein each of these components
is found at the ratio produced by the source. A naturally occurring
composition is one that is produced by an organism unmodified with
respect to the cellulolytic enzymes such that the ratio of the
component enzymes is unaltered from that produced by the native
organism.
[0045] A "non-naturally occurring" composition encompasses those
compositions produced by: (1) combining component cellulolytic
enzymes either in a naturally occurring ratio or non-naturally
occurring, i.e., altered, ratio; or (2) modifying an organism to
overexpress or underexpress one or more cellulolytic enzyme; or (3)
modifying an organism such that at least one cellulolytic enzyme is
deleted.
[0046] The whole cellulase mixtures useful in the present invention
may have one or more of the various EGs and/or CBHs deleted. For
example, EG1 may be deleted alone or in combination with other EGs
and/or CBHs. BGs may be over-expressed relative to the native
levels. Heterologous expression of BGs is also contemplated
herein.
[0047] "Carbon limitation" is a state wherein a microorganism has
just enough carbon to produce a desired protein product, but not
enough carbon to completely satisfy the organism's requirement,
e.g., sustain growth. Therefore, the maximal amount of carbon goes
toward protein production.
[0048] As used herein, the terms "promoter" and "cellulase
promoter" refers to a nucleic acid sequence that functions to
direct transcription of a downstream gene and are used
interchangeably herein. The promoter will generally be appropriate
to the host cell in which the target gene is being expressed. The
promoter together with other transcriptional and translational
regulatory nucleic acid sequences (also termed "control sequences")
are necessary to express a given gene. In general, the
transcriptional and translational regulatory sequences include, but
are not limited to, promoter sequences, ribosomal binding sites,
transcriptional start and stop sequences, translational start and
stop sequences, and enhancer or activator sequences. In one aspect
the promoter is an inducible promoter. In another aspect the
promoter is inducible by an inducer selected from the group
consisting of gentiobiose, cellulose and sophorose. In one aspect
the promoter is the T. reesei cbh1 promoter which is deposited in
GenBank under Accession Number D86235. In another aspect the
promoter is a cbh II or xylanase promoter from T. reesei.
[0049] As used herein, a "promotor sequence" is a DNA sequence
which is recognized by the particular filamentous fungus for
expression purposes. A "promoter" is defined as an array of nucleic
acid control sequences that direct transcription of a nucleic acid.
As used herein, a promoter includes necessary nucleic acid
sequences near the start site of transcription, such as, in the
case of a polymerase II type promoter, a TATA element. A
"constitutive" promoter is a promoter that is active under most
environmental and developmental conditions. An "inducible" promoter
is a promoter that is active under environmental or developmental
regulation. An example of an inducible promoter useful in the
present invention is the T. reesei cbh 1 promoter. The term
"operably linked" refers to a functional linkage between a nucleic
acid expression control sequence (such as a promoter, or array of
transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence directs
transcription of the nucleic acid corresponding to the second
sequence.
[0050] Examples include the promoter from the A. awamorior A. niger
glucoamylase genes (Nunberg, J. H. et al. (1984) Mol. Cell. Biol.
4, 2306-2315; Boel, E. et al. (1984) EMBO J. 3, 1581-1585), the
Mucor miehei carboxyl protease gene herein, the Trichoderma reesei
cellobiohydrolase I gene (Shoemaker, S. P. et al. (1984) European
Patent Application No. EPO0137280A1), the A. nidulans trpC gene
(Yelton, M. et al. (1984) Proc. Natl. Acad. Sci. USA 81,1470-1474;
Mullaney, E. J. et al. (1985) Mol. Gen. Genet. 199, 37-45) the A.
nidulans alcA gene (Lockington, R. A. et al. (1986) Gene 33,
137-149), the A. nidulans tpiA gene (McKnight, G. L. et al. (1986)
Cell 46, 143-147), the A. nidulans amdS gene (Hynes, M. J. et al.
(1983) Mol. Cell Biol. 3,1430-1439), the T. reesei xln1 gene, the
T. reesei cbh2 gene, the T. reesei eg1 gene, the T. reesei eg2
gene, the T. reesei eg3 gene, and higher eukaryotic promoters such
as the SV40 early promoter (Barclay, S. L. and E. Meller (1983)
Molecular and Cellular Biology 3, 2117-2130).
[0051] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA encoding a secretory leader, i.e., a signal peptide,
is operably linked to DNA for a polypeptide if it is expressed as a
preprotein that participates in the secretion of the polypeptide; a
promoter or enhancer is operably linked to a coding sequence if it
affects the transcription of the sequence; or a ribosome binding
site is operably linked to a coding sequence if it is positioned so
as to facilitate translation. Generally, "operably linked" means
that the DNA sequences being linked are contiguous, and, in the
case of a secretory leader, contiguous and in reading phase.
However, enhancers do not have to be contiguous. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist, the synthetic oligonucleotide adaptors or
linkers are used in accordance with conventional practice.
[0052] As used herein, the term "gene" means the segment of DNA
involved in producing a polypeptide chain, that may or may not
include regions preceding and following the coding region, e.g. 5'
untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer"
sequences, as well as intervening sequences (introns) between
individual coding segments (exons).
[0053] The gene may encode therapeutically significant proteins or
peptides, such as growth factors, cytokines, ligands, receptors and
inhibitors, as well as vaccines and antibodies. The gene may encode
commercially important industrial proteins or peptides, such as
enzymes, e.g., proteases, mannanases, xylanases, amylases,
glucoamylases, cellulases, oxidases and lipases. The gene of
interest may be a naturally occurring gene, a mutated gene or a
synthetic gene.
[0054] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all.
[0055] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger peptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0056] "Induction" refers to the increased transcription of a gene
resulting in the synthesis of a protein of interest in a cell or
organism at a markedly increased rate in response to the presence
of an "inducer". To measure the induction of a protein of interest,
cells treated with a potential inducer are compared to control
samples without the inducer. Control samples (untreated with
inducers) are assigned a relative protein activity value of 100%.
Induction of a polypeptide is achieved when the activity value
relative to the control (untreated with inducers) is greater than
100%, greater than 110%, more preferably 150%, more preferably
200-500% (i.e., two to five fold higher relative to the control),
or more preferably 1000-3000% higher.
[0057] The "filamentous fungi" of the present invention are
eukaryotic microorganisms and include all filamentous forms of the
subdivision Eumycotina (see Alexopoulos, C. J. (1962), Introductory
Mycology, New York: Wiley). These fungi are characterized by a
vegetative mycelium with a cell wall composed of chitin, cellulose,
and other complex polysaccharides. The filamentous fungi of the
present invention are morphologically, physiologically, and
genetically distinct from yeasts. Vegetative growth by filamentous
fungi is by hyphal elongation and carbon catabolism is obligately
aerobic. In contrast, vegetative growth by yeasts such as S.
cerevisiae is by budding of a unicellular thallus, and carbon
catabolism may be fermentative. S. cerevisiae has a prominent, very
stable diploid phase, whereas diploids exist only briefly prior to
meiosis in filamentous fungi, e.g., Aspergillus and Neurospora. S.
cervisiae has 17 chromosomes as opposed to 8 and 7 for A. nidulans
and N. crassa respectively. Recent illustrations of differences
between S. cerevisiae and filamentous fungi include the inability
of S. cerevisiae to process Aspergillus and Trichoderma introns and
the inability to recognize many transcriptional regulators of
filamentous fungi (Innis, M. A. et al. (1985) Science,
228,21-26).
[0058] "Glucosidases" refers to any enzyme whose end product is
glucose.
[0059] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not normally found in the same
relationship to each other in nature. For instance, the nucleic
acid is typically recombinantly produced, having two or more
sequences, e.g., from unrelated genes arranged to make a new
functional nucleic acid, e.g., a promoter from one source and a
coding region from another source. Similarly, a heterologous
protein will often refer to two or more subsequences that are not
found in the same relationship to each other in nature (e.g., a
fusion protein).
[0060] An "incubation product" refers to a solution that was held
or incubated at an elevated temperature for a specific period of
time.
[0061] An "inducer" is any compound that causes cells to produce
larger amounts of enzymes or other substances than they would
otherwise produce if the inducer was absent.
[0062] "Inducing feed", refers to a solution fed to a microorganism
that causes or induces the production of the desired protein
product.
[0063] The terms "isolated" or "purified" as used herein refer to a
nucleic acid or amino acid that is removed from at least one
component with which it is naturally associated.
II. Protein of Interest or Desired Protein
[0064] The terms protein of interest and desired protein may be
used interchangeably herein. The present invention is particularly
useful in enhancing the intracellular and/or extracellular
production of proteins. The protein may be homologous or
heterologous. Proteins that may produced by the instant invention
include, but are not limited to, hormones, enzymes, growth factors,
cytokines, antibodies and the like.
[0065] Hormones include, but are not limited to,
follicle-stimulating hormone, luteinizing hormone,
corticotropin-releasing factor, somatostatin, gonadotropin hormone,
vasopressin, oxytocin, erythropoietin, insulin and the like.
[0066] Growth factors are proteins that bind to receptors on the
cell surface, with the primary result of activating cellular
proliferation and/or differentiation. Growth factors include, but
are not limited to, platelet-derived growth factor, epidermal
growth factor, nerve growth factor, fibroblast growth factors,
insulin-like growth factors, transforming growth factors and the
like.
[0067] Cytokines are a unique family of growth factors. Secreted
primarily from leukocytes, cytokines stimulate both the humoral and
cellular immune responses, as well as the activation of phagocytic
cells. Cytokines include, but are not limited to, colony
stimulating factors, the interleukins (IL-1 (.alpha. and .beta.),
IL-2 through IL-13) and the interferons (.alpha., .beta. and
.gamma.).
[0068] Human Interleukin-3 (IL-3) is a 15 kDa protein containing
133 amino acid residues. IL-3 is a species specific colony
stimulating factor which stimulates colony formation of
megakaryocytes, neutrophils, and macrophages from bone marrow
cultures.
[0069] Antibodies include, but are not limited to, immunoglobulins
from any species from which it is desirable to produce large
quantities. It is especially preferred that the antibodies are
human antibodies. Immunoglobulins may be from any class, i.e., G,
A, M, E or D.
[0070] Additionally, a "protein of interest" or "polypeptide of
interest" refers to the protein to be expressed and secreted by the
host cell. The protein of interest may be any protein that up until
now has been considered for expression in prokaryotes. In one
embodiment, the protein of interest which is expressed and secreted
include proteins comprising a signal peptide. The protein of
interest may be either homologous or heterologous to the host.
Thus, a protein of interest may be a secreted polypeptide
particularly an enzyme which is selected from amylolytic enzymes,
proteolytic enzymes, cellulolytic enzymes, oxido-reductase enzymes
and plant wall degrading enzymes. Examples of these enzymes include
amylases, proteases, xylanases, lipases, laccases, phenol oxidases,
oxidases, cutinases, cellulases, hemicellulases, esterases,
perioxidases, catalases, glucose oxidases, phytases, pectinases,
glucosidases, isomerases, transferases, galactosidases and
chitinases. The secreted polypeptide may also be a hormone, a
growth factor, a receptor, vaccine, antibody or the like. In an
embodiment the secreted polypeptide is a cellulolytic enzyme.
III. Molecular Biology
[0071] In one embodiment this invention provides for the expression
of heterologous genes under control of the cellulase gene promoters
of Trichoderma reesei. Therefore, this invention relies on routine
techniques in the field of recombinant genetics. Basic texts
disclosing the general methods of use in this invention include
Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.
1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual
(1990); and Ausubel et al., eds., Current Protocols in Molecular
Biology (1994)).
[0072] Heterologous genes comprising the cellulase gene promoter
sequences of filamentous fungi are typically cloned into
intermediate vectors before transformation into Trichoderma reesei
cells for replication and/or expression. These intermediate vectors
are typically prokaryotic vectors, e.g., plasmids, or shuttle
vectors.
[0073] To obtain high level expression of a cloned gene, the
heterologous gene is preferably positioned about the same distance
from the promoter as is in the naturally occurring cellulase gene.
As is known in the art, however, some variation in this distance
can be accommodated without loss of promoter function.
[0074] Those skilled in the art are aware that a natural promoter
can be modified by replacement, substitution, addition or
elimination of one or more nucleotides without changing its
function. The practice of the invention encompasses and is not
constrained by such alterations to the promoter.
[0075] The expression vector/construct typically contains a
transcription unit or expression cassette that contains all the
additional elements required for the expression of the heterologous
sequence. A typical expression cassette thus contains a promoter
operably linked to the heterologous nucleic acid sequence and
signals required for efficient polyadenylation of the transcript,
ribosome binding sites, and translation termination. Additional
elements of the cassette may include enhancers and, if genomic DNA
is used as the structural gene, introns with functional splice
donor and acceptor sites.
[0076] The practice of the invention is not constrained by the
choice of promoter in the genetic construct. However, exemplary
promoters are the Trichoderma reesei cbh1, cbh2, eg1, eg2, eg3,
eg5, xIn1 and xIn2 promoters.
[0077] In addition to a promoter sequence, the expression cassette
should also contain a transcription termination region downstream
of the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes.
[0078] Although any fungal terminator is likely to be functional in
the present invention, preferred terminators include: the
terminator from Aspergillus nidulans trpC gene (Yelton, M. et al.
(1984) PNAS USA 81:1470-1474, Mullaney, E. J. et al. (1985) MGG
199:37-45), the Aspergillus awamori or Aspergillus niger
glucoamylase genes (Nunberg, J. H. et al. (1984) Mol. Cell Biol.
4:2306, Boel, E. et al. (1984) EMBO J. 3:1581-1585) and the Mucor
miehei carboxyl protease gene (EPO Publication No. 0 215 594).
[0079] The particular expression vector used to transport the
genetic information into the cell is not particularly critical. Any
of the conventional vectors used for expression in eukaryotic or
prokaryotic cells may be used. Standard bacterial expression
vectors include bacteriophages .lamda. and M13, as well as plasmids
such as pBR322 based plasmids, pSKF, pET23D, and fusion expression
systems such as MBP, GST, and LacZ. Epitope tags can also be added
to recombinant proteins to provide convenient methods of isolation,
e.g., c-myc.
[0080] The elements that are typically included in expression
vectors also include a replicon, a gene encoding antibiotic
resistance to permit selection of bacteria that harbor recombinant
plasmids, and unique restriction sites in nonessential regions of
the plasmid to allow insertion of heterologous sequences. The
particular antibiotic resistance gene chosen is not critical, any
of the many resistance genes known in the art are suitable. The
prokaryotic sequences are preferably chosen such that they do not
interfere with the replication or integration of the DNA in
Trichoderma reesei.
[0081] The methods of transformation of the present invention may
result in the stable integration of all or part of the
transformation vector into the genome of the filamentous fungus.
However, transformation resulting in the maintenance of a
self-replicating extra-chromosomal transformation vector is also
contemplated.
[0082] Many standard transfection methods can be used to produce
Trichoderma reesei cell lines that express large quantities of the
heterologus protein. Some of the published methods for the
introduction of DNA constructs into cellulase-producing strains of
Trichoderma include Lorito, Hayes, DiPietro and Harman, 1993, Curr.
Genet. 24: 349-356; Goldman, VanMontagu and Herrera-Estrella, 1990,
Curr. Genet. 17:169-174; Penttila, Nevalainen, Ratto, Salminen and
Knowles, 1987, Gene 6: 155-164, for Aspergillus Yelton, Hamer and
Timberlake, 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, for
Fusarium Bajar, Podila and Kolattukudy, 1991, Proc. Natl. Acad.
Sci. USA 88: 8202-8212, for Streptomyces Hopwood et al., 1985, The
John Innes Foundation, Norwich, UK and for Bacillus Brigidi,
DeRossi, Bertarini, Riccardi and Matteuzzi, 1990, FEMS Microbiol.
Lett. 55: 135-138).
[0083] However, any of the well-known procedures for introducing
foreign nucleotide sequences into host cells may be used. These
include the use of calcium phosphate transfection, polybrene,
protoplast fusion, electroporation, biolistics, liposomes,
microinjection, plasma vectors, viral vectors and any of the other
well known methods for introducing cloned genomic DNA, cDNA,
synthetic DNA or other foreign genetic material into a host cell
(see, e.g., Sambrook et al., supra). Also of use is the
Agrobacterium-mediated transfection method described in U.S. Pat.
No. 6,255,115. It is only necessary that the particular genetic
engineering procedure used be capable of successfully introducing
at least one gene into the host cell capable of expressing the
heterologous gene.
[0084] After the expression vector is introduced into the cells,
the transfected cells are cultured under conditions favoring
expression of genes under control of cellulase gene promoter
sequences. Large batches of transformed cells can be cultured as
described below. Finally, product is recovered from the culture
using standard techniques.
[0085] Thus, the invention herein provides for the expression and
enhanced secretion of desired polypeptides whose expression is
under control of cellulase gene promoter sequences including
naturally occurring cellulase genes, fusion DNA sequences, and
various heterologous constructs. The invention also provides
processes for expressing and secreting high levels of such desired
polypeptides.
IV. Filamentous Fungi
[0086] Filamentous fungi include all filamentous forms of the
subdivision Eumycota and Oomycota. The filamentous fungi are
characterized by vegetative mycelium having a cell wall composed of
chitin, glucan, chitosan, mannan, and other complex
polysaccharides, with vegetative growth by hyphal elongation and
carbon catabolism that is obligately aerobic.
[0087] In the present invention, the filamentous fungal parent cell
may be a cell of a species of, but not limited to, Trichoderma,
e.g., Trichoderma longibrachiatum (reesei), Trichoderma viride,
Trichoderma koningii, Trichoderma harzianum; Penicillium sp.;
Humicola sp., including Humicola insolens; Chrysosporium sp.,
including C. lucknowense; Gliocladium sp.; Aspergillus sp.;
Fusarium sp., Neurospora sp., Hypocrea sp., and Emericella sp. As
used herein, the term "Trichoderma" or "Trichoderma sp." refers to
any fungal strains which have previously been classified as
Trichoderma or are currently classified as Trichoderma.
[0088] In one preferred embodiment, the filamentous fungal parent
cell is an Aspergillus niger, Aspergillus awamori, Aspergillus
aculeatus, or Aspergillus nidulans cell.
[0089] In another preferred embodiment, the filamentous fungal
parent cell is a Trichoderma reesei cell.
V. Protein Expression
[0090] Proteins of the present invention are produced by culturing
cells transformed with an expression vector containing genes whose
expression is under control of cellulase gene promoter sequences.
The present invention is particularly useful for enhancing the
intracellular and/or extracellular production of proteins. The
protein may be homologous or heterologous. Proteins that may
produced by the instant invention include, but are not limited to,
hormones, enzymes, growth factors, cytokines, antibodies and the
like.
[0091] Enzymes include, but are not limited to, hydrolases, such as
protease, esterase, lipase, phenol oxidase, permease, amylase,
pullulanase, xylanase, cellulase, glucose isomerase, laccase and
protein disulfide isomerase.
[0092] Hormones include, but are not limited to,
follicle-stimulating hormone, luteinizing hormone,
corticotropin-releasing factor, somatostatin, gonadotropin hormone,
vasopressin, oxytocin, erythropoietin, insulin and the like.
[0093] Growth factors are proteins that bind to receptors on the
cell surface, with the primary result of activating cellular
proliferation and/or differentiation. Growth factors include, but
are not limited to, platelet-derived growth factor, epidermal
growth factor, nerve growth factor, fibroblast growth factors,
insulin-like growth factors, transforming growth factors and the
like.
[0094] Cytokines are a unique family of growth factors. Secreted
primarily from leukocytes, cytokines stimulate both the humoral and
cellular immune responses, as well as the activation of phagocytic
cells. Cytokines include, but are not limited to, colony
stimulating factors, the interleukins (IL-1 .alpha. and .beta.,
IL-2 through IL-13) and the interferons (.alpha., .beta. and
.gamma.).
[0095] Human Interleukin-3 (IL-3) is a 15 kDa protein containing
133 amino acid residues. IL-3 is a species specific colony
stimulating factor which stimulates colony formation of
megakaryocytes, neutrophils, and macrophages from bone marrow
cultures.
[0096] Antibodies include, but are not limited to, immunoglobulins
from any species from which it is desirable to produce large
quantities. It is especially preferred that the antibodies are
human antibodies. Immunoglobulins may be from any class, i.e., IgG,
IgM, IgA, IgD or IgE.
[0097] Proteins of interest in the present invention may also be
modified in a way to form chimeric molecules comprising a protein
of interest fused to another, heterologous polypeptide or amino
acid sequence. In one embodiment, such a chimeric molecule
comprises a fusion of the protein of interest with a tag
polypeptide which provides an epitope to which an anti-tag antibody
can selectively bind. The epitope tag is generally placed at the
amino-or carboxyl-terminus of the protein of interest.
[0098] Various tag polypeptides and their respective antibodies are
well known in the art. Examples include poly-histidine (poly-his)
or poly-histidine-glycine (poly-his-gly) tags; HIS6 and metal
chelation tags, the flu HA tag polypeptide and its antibody 12CA5
(Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)); the c-myc tag
and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et
al., Molecular and Cellular Biology 5:3610-3616 (1985)); and the
Herpes Simplex virus glycoprotein D (gD) tag and its antibody
(Paborsky et al., Protein Engineering 3(6):547-553 (1990)). Other
tag polypeptides include the FLAG-peptide (Hopp et al.,
BioTechnology 6:1204-1210 (1988)); the KT3 epitope peptide (Martin
et al., Science 255:192-194 (1992)); tubulin epitope peptide
(Skinner et al., J. Biol. Chem. 266:15163-15166 (1991)); and the T7
gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA 87:6393-6397 (1990)).
[0099] In an alternative embodiment, the chimeric molecule may
comprise a fusion of a protein of interest with an immunoglobulin
or a particular region of an immunoglobulin. For a bivalent form of
the chimeric molecule, such a fusion could be to the Fc region of
an IgG molecule.
[0100] Conditions appropriate for expression of said genes comprise
providing to the culture an inducing feed composition of the
instant invention. Optimal conditions for the production of the
proteins will vary with the choice of the host cell, and with the
choice of protein to be expressed. Such conditions will be easily
ascertained by one skilled in the art through routine
experimentation or optimization.
[0101] The protein of interest is typically purified or isolated
after expression. The protein of interest may be isolated or
purified in a variety of ways known to those skilled in the art
depending on what other components are present in the sample.
Standard purification methods include electrophoretic, molecular,
immunological and chromatographic techniques, including ion
exchange, hydrophobic, affinity, and reverse-phase HPLC
chromatography, and chromatofocusing. For example, the protein of
interest may be purified using a standard anti-protein of interest
antibody column. Ultrafiltration and diafiltration techniques, in
conjunction with protein concentration, are also useful. For
general guidance in suitable purification techniques, see Scopes,
Protein Purification (1982). The degree of purification necessary
will vary depending on the use of the protein of interest. In some
instances no purification will be necessary.
VI. Fermentation
[0102] The invention relies on fermentation procedures for
culturing fungi and bacteria. Fermentation procedures for
production of cellulase enzymes are known per se in the art. For
example, cellulase enzymes can be produced either by solid or
submerged culture, including batch, fed-batch and continuous-flow
processes.
[0103] Culturing is accomplished in a growth medium comprising an
aqueous mineral salts medium, organic growth factors, the carbon
and energy source material, molecular oxygen, and, of course, a
starting inoculum of one or more particular microorganism species
to be employed.
[0104] In addition to the carbon and energy source, oxygen,
assimilable nitrogen, and an inoculum of the microorganism, it is
necessary to supply suitable amounts in proper proportions of
mineral nutrients to assure proper microorganism growth, maximize
the assimilation of the carbon and energy source by the cells in
the microbial conversion process, and achieve maximum cellular
yields with maximum cell density in the fermentation media.
[0105] The composition of the aqueous mineral medium can vary over
a wide range, depending in part on the microorganism and substrate
employed, as is known in the art. The mineral media should include,
in addition to nitrogen, suitable amounts of phosphorus, magnesium,
calcium, potassium, sulfur, and sodium, in suitable soluble
assimilable ionic and combined forms, and also present preferably
should be certain trace elements such as copper, manganese,
molybdenum, zinc, iron, boron, and iodine, and others, again in
suitable soluble assimilable form, all as known in the art.
[0106] The fermentation reaction is an aerobic process in which the
molecular oxygen needed is supplied by a molecular
oxygen-containing gas such as air, oxygen-enriched air, or even
substantially pure molecular oxygen, provided to maintain the
contents of the fermentation vessel with a suitable oxygen partial
pressure effective in assisting the microorganism species to grow
in a thriving fashion. In effect, by using an oxygenated
hydrocarbon substrate, the oxygen requirement for growth of the
microorganism is reduced. Nevertheless, molecular oxygen must be
supplied for growth, since the assimilation of the substrate and
corresponding growth of the microorganisms, is, in part, a
combustion process.
[0107] Although the aeration rate can vary over a considerable
range, aeration generally is conducted at a rate which is in the
range of about 0.5 to 10, preferably about 0.5 to 7, volumes (at
the pressure employed and at 25.degree. C.) of oxygen-containing
gas per liquid volume in the fermentor per minute. This amount is
based on air of normal oxygen content being supplied to the
reactor, and in terms of pure oxygen the respective ranges would be
about 0.1 to 1.7, or preferably about 0.1 to 1.3, volumes (at the
pressure employed and at 25.degree. C.) of oxygen per liquid volume
in the fermentor per minute.
[0108] The pressure employed for the microbial conversion process
can range widely. Pressures generally are within the range of about
0 to 50 psig, presently preferably about 0 to 30 psig, more
preferably at least slightly over atmospheric pressure, as a
balance of equipment and operating cost versus oxygen solubility
achieved. Greater than atmospheric pressures are advantageous in
that such pressures do tend to increase a dissolved oxygen
concentration in the aqueous ferment, which in turn can help
increase cellular growth rates. At the same time this is balanced
by the fact that high atmospheric pressures do increase equipment
and operating costs.
[0109] The fermentation temperature can vary somewhat, but for
filamentous fungi such as Trichoderma reesei the temperature
generally will be within the range of about 20.degree. C. to
40.degree. C., generally preferably in the range of about
25.degree. C. to 34.degree. C., depending on the strain of
microorganism chosen.
[0110] The microorganisms also require a source of assimilable
nitrogen. The source of assimilable nitrogen can be any
nitrogen-containing compound or compounds capable of releasing
nitrogen in a form suitable for metabolic utilization by the
microorganism. While a variety of organic nitrogen source
compounds, such as protein hydrolysates, can be employed, usually
cheap nitrogen-containing compounds such as ammonia, ammonium
hydroxide, urea, and various ammonium salts such as ammonium
phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium
chloride, or various other ammonium compounds can be utilized.
Ammonia gas itself is convenient for large scale operations, and
can be employed by bubbling through the aqueous ferment
(fermentation medium) in suitable amounts. At the same time, such
ammonia can also be employed to assist in pH control.
[0111] The pH range in the aqueous microbial ferment (fermentation
admixture) should be in the exemplary range of about 2.0 to 8.0.
With filamentous fungi, the pH normally is within the range of
about 2.5 to 8.0; with Trichoderma reesei, the pH normally is
within the range of about 3.0 to 7.0. pH range preferences for
certain microorganisms are dependent on the media employed to some
extent, as well as the particular microorganism, and thus change
somewhat with change in media as can be readily determined by those
skilled in the art.
[0112] While the average retention time of the fermentation
admixture in the fermentor can vary considerably, depending in part
on the fermentation temperature and culture employed, generally it
will be within the range of about 24 to 500 hours, preferably
presently about 24 to 400 hours.
[0113] Preferably, the fermentation is conducted in such a manner
that the carbon-containing substrate can be controlled as a
limiting factor, thereby providing good conversion of the
carbon-containing substrate to cells and avoiding contamination of
the cells with a substantial amount of unconverted substrate. The
latter is not a problem with water-soluble substrates, since any
remaining traces are readily washed off. It may be a problem,
however, in the case of non-water-soluble substrates, and require
added product-treatment steps such as suitable washing steps.
[0114] As described above, the time to reach this level is not
critical and may vary with the particular microorganism and
fermentation process being conducted. However, it is well known in
the art how to determine the carbon source concentration in the
fermentation medium and whether or not the desired level of carbon
source has been achieved.
[0115] Although the fermentation can be conducted as a batch or
continuous operation, fed batch operation is much to be preferred
for ease of control, production of uniform quantities of products,
and most economical uses of all equipment.
[0116] If desired, part or all of the carbon and energy source
material and/or part of the assimilable nitrogen source such as
ammonia can be added to the aqueous mineral medium prior to feeding
the aqueous mineral medium to the fermentor.
[0117] Each of the streams introduced into the reactor preferably
is controlled at a predetermined rate, or in response to a need
determinable by monitoring such as concentration of the carbon and
energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide in
the off-gases from the fermentor, cell density measurable by light
transmittancy, or the like. The feed rates of the various materials
can be varied so as to obtain as rapid a cell growth rate as
possible, consistent with efficient utilization of the carbon and
energy source, to obtain as high a yield of microorganism cells
relative to substrate charge as possible.
[0118] In either a batch, or the preferred fed batch operation, all
equipment, reactor, or fermentation means, vessel or container,
piping, attendant circulating or cooling devices, and the like, are
initially sterilized, usually by employing steam such as at about
121.degree. C. for at least about 15 minutes. The sterilized
reactor then is inoculated with a culture of the selected
microorganism in the presence of all the required nutrients,
including oxygen, and the carbon-containing substrate. The type of
fermentor employed is not critical, though presently preferred is
operation under 15 L Biolafitte (Saint-Germain-en-Laye,
France).
[0119] The collection and purification of the cellulose enzymes
from the fermentation broth can also be done by procedures known
per se in the art. The fermentation broth will generally contain
cellular debris, including cells, various suspended solids and
other biomass contaminants, as well as the desired cellulase enzyme
product, which are preferably removed from the fermentation broth
by means known in the art.
[0120] Suitable processes for such removal include conventional
solid-liquid separation techniques such as, e.g., centrifugation,
filtration, dialysis, microfiltration, rotary vacuum filtration, or
other known processes, to produce a cell-free filtrate. It may be
preferable to further concentrate the fermentation broth or the
cell-free filtrate prior to crystallization using techniques such
as ultrafiltration, evaporation or precipitation.
[0121] Precipitating the proteinaceous components of the
supernatant or filtrate may be accomplished by means of a salt,
e.g., ammonium sulfate, followed by purification by a variety of
chromatographic procedures, e.g., ion exchange chromatography,
affinity chromatography or similar art recognized procedures.
Examples
[0122] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
[0123] This example illustrates how an inducing feed composition
for stimulating the expression of cellulase genes in Trichoderma
reesei was prepared. The incubation was run at the pH of the
solution, i.e., 5.0. For beta-glucosidase the incubation was found
to be best at pH 4.0-6.5.
[0124] (i) A 60% (w/w) glucose solution was sterilized for 30
minutes at 121.degree. C., 2.2 bar pressure.
[0125] (ii) Sterile whole cellulase preparation was added to the
glucose solution to a final concentration of 1 0 g total
protein/L.
[0126] (iii) The tank containing the glucose and whole cellulase
mixture was held at 65.degree. C. for 3 days with 75 RPM
mixing.
[0127] (iv) Following incubation, the sterile solution was
harvested to an appropriate container for fermentation feeding.
[0128] The resulting inducing feed composition was found to have
16.1 g/L Sophorose, 47.5 g/L Gentiobiose, and approximately 600 g/L
Glucose. Other sugars may be present but were not analyzed.
[0129] Inducing feed solutions have also been prepared from
solutions of 20% and 60% glucose. The higher the glucose solution,
the higher the final sophorose concentration.
[0130] Whole cellulase preparation has been used at final
concentrations of 2 g and 10 g total protein/L. The higher the
protein loading, the higher the ending sophorose concentration. See
FIG. 6. Ultimately however, it is expected that a longer reaction
at the lower concentration of whole cellulase preparation will
achieve the same sophorose levels if the solution is incubated for
a longer period of time.
[0131] The incubation temperature also influences sophorose
production. For example, sophorose concentration was 2 times as
high when the composition was incubated at 65.degree. C. than when
the composition was incubated at 50.degree. C.
Example 2
[0132] The following example details how a glucose/sophorose feed
is made and used to produce cellulase enzyme during
fermentation.
I. Production of Glucose/Sophorose Feed:
[0133] 60% (w/w) glucose solution was dissolved and sterilized for
30 minutes at 121.degree. C. The temperature was decreased to
65.degree. C. and 10 g of total protein (whole cellulase previously
produced by T. reesei)/L was added. The mixture was agitated slowly
and held at 65.degree. C. for 3 days. The sophorose content was
measured at 12 g/L in this 60% glucose solution.
II. Fermentation
[0134] 0.8 L of media was inoculated with 1.5 ml Trichoderma reesei
RL-P37 frozen spore suspension as a seed flask. This flask was
split into two 0.4 L portions and transferred to 2.times.7 L of
fermentation media in two different 15 L Biolafitte fermentors
after 48 hours. The growth media had the following composition:
TABLE-US-00001 Media component g/L KH.sub.2PO.sub.4 4
(NH.sub.4)2SO.sub.4 6.35 MgSO.sub.4--7H.sub.2O 2
CaCl.sub.2--2H.sub.2O 0.53 Glucose 50 Corn Steep Solids 6.25
(Roquette) Trace elements* 1 ml/L Trace elements*: 5 g/L
FeSO.sub.4--7H.sub.2O; 1.6 g/L MnSO.sub.4--H.sub.2O; 1.4 g/L
ZnSO.sub.4--7H.sub.2O.
[0135] The fermentor was run at 25.degree. C., 750 RPM and 8
standard liters per minute (SLM) airflow.
[0136] The glucose/sophorose was added in place of glucose in the
batch phase for the experimental tank but pure glucose was used in
the control. This batched glucose was exhausted at approximately 20
hours at which point the cells stopped growing and a carbon
limiting feed was begun. A 40% glucose/sophorose feed was added at
0.25 g/minute with 40% pure glucose solution being fed to the
control tank (diluted from feed formation detailed above). Total
protein, which is directly correlated with cellulase production
(based upon our comparison of total extracellular protein vs
cellulase activity), was induced just after the batch phase in the
glucose/sophorose tank but not in the glucose control tank. Thus,
pretreatment of the glucose with whole cellulase is required to
produce cellulase on glucose with Trichoderma reesei RL-P37. See
FIG. 1.
Example 3
[0137] The following example details how a glucose/sophorose feed
is made and used to produce a heterologous protein from a
filamentous fungus during fermentation.
[0138] The inducing feed composition is prepared using the
procedure in Example 1.
[0139] An expression plasmid for use in transforming Trichoderma
reesei is constructed as follows. The ends of the gene encoding
protein of interest are blunted by T4 DNA polymerase and inserted
into Pmel restriction site of the Trichoderma expression vector,
pTEX, see PCT Publication No. WO 96/23928, which publication is
herein incorporated by reference, which contains a CBH1 promoter
and terminator for gene expression and a Trichoderma pyr4 gene as a
selection marker for transformants. The linear DNA fragment
containing only the CBH1 promoter, the gene encoding the protein of
interest, the CBH1 terminator and selection marker pyr4 is isolated
from a gel and used to transform a uridine auxotroph strain of
Trichoderma reesei (see U.S. Pat. No. 5,472,864) which has the four
major cellulase genes deleted. Stable transformants are isolated on
Trichoderma minimal plates without uridine. The transformants are
grown on 50 ml of Proflo medium in shake flasks for 4 days at
28.degree. C. to 30.degree. C. and expression of the protein of
interest is assayed by methods known to one skilled in the art.
Proflo medium is composed of (g/l) Proflo 22.5; lactose 30.0;
(NH.sub.4).sub.2SO.sub.4 6.5 KH.sub.2PO.sub.4 2.0; MgSO.sub.47
H.sub.2O 0.3; CaCL.sub.2 0.2; CaC0.sub.3 0.72; trace metal stock
solution 1.0 ml/l and 10% Tween 80 2.0 ml/l. The trace metal stock
solution used had (g/l) FeSO.sub.4.7H.sub.2O 5.0;
MnSO.sub.4.H.sub.2O 1.6; ZnSO.sub.4.7H.sub.2O 1.4;
CoCl.sub.2.6H.sub.2O) 2.8.
[0140] The shake flasks are divided and placed in a 15 L fermentor
as described in Example 2. Expression of the protein of interest is
induced by the inducing feed composition but not the glucose
solution.
Example 4
[0141] This example details how the enzyme may be immobilized for
the production of an inducing feed solution.
[0142] A whole cellulase broth comprising a .beta.-glucosidase is
immobilized according to the method described in U.S. Pat. No.
5,541,097. Briefly, 10 gm of bentonite were in 500 ml water to
which 11 ml of 10% PEI was added. Separately 20 ml of whole
cellulase (200 g Total Protein/L) was added to 250 ml of 0.02 M
acetate buffer at pH 5.5. Then 4.44 ml of 50% Glutaraldehyde
(Fischer, Reagent Grade) was added to the enzyme solution while the
pH was maintained at 5.5. After 2 hours the enzyme complex was
added to the bentonite complex giving a total volume of about 750
ml. This mixture was mixed overnight at 4.degree. C. The complex
was then collected on a Buchner funnel and washed with a large
quantity of water. The cake was then resuspended in 0.02 M acetate
buffer, with a final weight of 175 gm.
[0143] It is difficult to quantitate the enzyme activity remaining
after immobilization because whole cellulase contains greater than
five different enzymes, each with different activities (the
immobilized enzyme was shown to reduce the viscosity of barley
flour slurry, so cellulase activity was known to be present).
Therefore, cellulase loading was done based upon how much enzyme
was immobilized, not how much remained active. The final slurry was
determined to contain 0.022 g total protein/ g slurry.
[0144] Sophorose production from the immobilized cellulase was
examined at two different enzyme loading and glucose
concentrations: [0145] 1) 23 g slurry+29.5 ml 67% (w/w) glucose=10
g/L protein loading@40.7% glucose [0146] 2) 7.6 g slurry+44.8 ml
67% (w/w) glucose=3.2 g/L protein loading@60% glucose Each 52.5 ml
volume was added to a 250 ml Erlenmeyer flask, agitated at 100 RPM
and incubated at 65.degree. C. over several days.
[0147] The rate of sophorose production for each of the two cases
was lower than the control case where the same amount of enzyme
solution was added as immobilized enzyme (FIGS. 2-3). This was not
surprising, as it is assumed that some of the enzyme activity is
lost as the enzyme is immobilized. However, the large gain for
immobilization is that the enzyme can be used to make multiple
batches of glucose/ sophorose feed. FIG. 4 shows that centrifuging
the immobilized enzyme out of the glucose solution and repeating
the experiment as shown above, the same final sophorose titers are
achieved in the first and second trials. This shows that the enzyme
is still active over at lease two uses, and likely over many more.
Therefore, even with some loss of enzyme activity from
immobilization, the ability to reuse the enzyme multiple times
makes cellulase immobilization the most attractive alternative for
glucose/ sophorose production.
Example 5
[0148] This example details how the production of an inducing feed
solution may be accomplished with the use of cellobiose as a
starting carbon source.
[0149] The experiments were run in the exact same way as the other
examples of sophorose production in shake flasks (50 ml in 250 ml
flask, 65.degree. C., 100 RPM) except for what is noted below. FIG.
5 compares the sophorose production in 25% cellobiose with that
produced in 25% glucose.
[0150] If sophorose is the "true" inducer in nature, it would most
likely have to be formed from cellobiose as T. reesei is very
unlikely to see even moderate levels of glucose in nature which
would be required to form sophorose via transglycosylation. FIG. 5
shows 25% (w/w) cellobiose incubated with 10 g/L cellulase compared
to 25% glucose. The sophorose produced from cellobiose peaked at
more than 10 g/L, three times the concentration that was produced
by glucose alone. However, that sophorose was then degraded down to
a similar level as that produced from glucose alone (4.1 g/L vs 2.5
g/L). This behavior seems to show the sophorose approaching an
equilibrium with the glucose, which was all that remained after all
of the cellobiose was cleaved around 29 hours.
[0151] It is highly unlikely that the .beta.-glucosidase enzyme
sees a sufficient concentration of glucose to form much sophorose
in nature. The cells are more likely to see high concentrations of
cellobiose, a breakdown product of cellulose. FIG. 5 shows that
three times as much sophorose is produced from cellobiose than from
glucose. The sophorose level appears to fall at the same time the
cellobiose is completely turned over to glucose and other
transglycosylation products at approximately 29 hours (cellobiose
data not shown). This would support a hypothesized mechanism where
the cellobiose is cleaved to two glucose molecules that then
rearrange and transglycosylate before leaving the active site. As
the cellobiose experiment continued, the rate of sophorose cleavage
was greater than the rate of sophorose formation from glucose
transglycosylation and the sophorose level fell to almost that of
glucose, 4.1 g/L compared to 2.5 g/L. This data strongly supports
the possibility that small amounts of sophorose are formed through
the cleavage of cellobiose by .beta.-glucosidase.
[0152] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *